The primary goal of the proposed studies is to establish whether precursor B-cell acute lymphoblastic leukemias (B-ALLs) that overexpress the cytokine receptor CRLF2 are dependent on CRLF2 signaling for survival. Using a functional screen, we recently identified CRLF2 as a driver of pro-growth signaling in approximately 15% of adult and high-risk pediatric B-ALL that lack common chromosomal rearrangements, and 60% of B-ALL in children with Down syndrome. Outcomes among adults with CRLF2-overexpressing B-ALL is particularly poor, with 0% 4-year overall survival in our series. CRLF2 overexpression uniformly results from genomic rearrangement of the CRLF2 locus, either through an interstitial deletion or a translocation. B- ALLs that overexpress CRLF2 fall into one of three categories, based on the presence or absence of somatic mutations within either CRLF2 itself or the receptor-associated kinase JAK2. Our central hypothesis is that B-ALLs with CRLF2 overexpression are dependent on CRLF2 signaling for survival. If so, these B-ALLs could be targeted with small-molecule kinase inhibitors, akin to the use of the kinase inhibitor imatinib for B-ALL that express the BCR/ABL fusion kinase. For Specific Aim #1, we will determine the role of mutant JAK2 in CRLF2 signaling. These studies will define the physical relationship between CRLF2 and JAK2 using lymphoid cell lines and purified proteins, determine whether that relationship depends on JAK2 mutation and clarify the essential regions of CRLF2 for JAK2 activity. In Aim #2, we will define the essential components of CRLF2 signaling, including members of the CRLF2 receptor complex, the receptor ligand, and downstream kinases. In Aim #3, we will define the dependence of B-ALL on CRLF2 signaling in vivo. These studies will use primary human xenografts and B-ALL cell lines to build on the findings from Aims #1 and #2, first by characterizing in vivo dependence on specific components of CRLF2 signaling, and then by correlating that dependence with the response to kinase inhibitors. The availability of therapeutic JAK inhibitors, which are now in clinical trials for the treatment of myeloproliferative disorders, has created an exciting opportunity to rapidly translate the discovery of CRLF2 into a directed therapy for patients with B-ALL. Finally, the primary xenografts will be an invaluable platform for our long-term efforts to define and overcome the biologic determinants of therapeutic resistance in this disease.